Memory B cells are responsible for faster, higher affinity, and more effective secondary humoral immune responses and are a fundamental cellular basis for long-term immunity to pathogens after initial exposure of vaccination. Although there is a fair degree of information about human memory cells, this system is not tenable for many types of experimentation and many of the conclusions are based on inferences and assumptions that have not and probably cannot be tested. On the other hand, although murine systems are open to experimentation, murine memory B cells are very poorly understood due to both a dearth of cells and a lack of useful markers to identify them, other than IgG. Our lab has developed systems to generate increased numbers of murine memory B cells and we have characterized them at the phenotypic and functional level. Notably, we have recently completed a gene expression comparison among naive, GC and memory B cells. Among differentially expressed genes, two with very substantial increases in expression in memory vs. naive cells LifR (75-fold) and BmpR11 (15-fold) are also of great conceptual interest because they are well-known to maintain homeostatic self-renewal capacity in pluripotent and multipotent stem cells. Our data, and that of others, is consistent with memory B cells having an extended half-life and undergoing slow homeostatic turnover, much like a stem cell. Memory cells differentiate from GC B cells, but the signals that control this key event are not known. Nor is it currently possible to reliably trace Ag- specific memory cells as they emerge from the GC and complete their development, lodging in follicles and the MZ. Both LifR and BmpR11 must be strongly induced at some juncture between the GC stage and the fully developed memory cell. Specifically, our hypothesis is that LifR and/or BmpR11 are turned on at or near the differentiation of memory cells from GC cells and may play a key role in lineage commitment and long-term survival. Moreover, high expression of these genes should mark memory B cells, providing the ready ability to identify them by FACS and histology. To test this hypothesis and to create highly useful systems to track, sort and transfer memory cells the essence of this proposal is to request funds to produce mice carrying fluorescent reporters under the control of these two loci. We will construct and validate these mice and then use them in initial studies to test basic hypotheses about how their expression correlates with memory B cell formation. The development of these novel strains is innovative in both the target genes being investigated as well as the approach. Aside from our having made the observations and insights concerning LifR and Bmpr1a, our lab is in an ideal position both to make these mice and also to properly evaluate and exploit them once constructed. As appropriate for an R21, perhaps most important are the windows that this work will open for future work and the potentially great value of these mice for investigations both within the immune system and in the stem cell field. PUBLIC HEALTH RELEVANCE: This work seeks to explore the role of two genes which we have recently discovered are massively increased in expression in memory B cells. Memory B cells form as a result of vaccination or infection and are very important in preventing reinfection. They are thought to live for long periods of time and are responsible for generating the cells that make antibody when there is exposure to a pathogen for the second or subsequent times. We propose to make novel mice that are engineered to express fluorescent proteins in any cells that are expressing these two genes. This will allow us to identify the cells and determine their properties, giving us insight into how and where memory B cells form.